U.S. patent number 5,985,600 [Application Number 08/411,859] was granted by the patent office on 1999-11-16 for nucleic acid encoding delta opioid receptor.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Robert H. Edwards, Christopher J. Evans, Duane E. Keith, Jr..
United States Patent |
5,985,600 |
Evans , et al. |
November 16, 1999 |
Nucleic acid encoding delta opioid receptor
Abstract
Nucleic acid molecules comprising a nucleotide sequence encoding
a mammalian delta opioid receptor are disclosed. The invention
provides recombinant materials for the production of mammalian
delta opioid receptors.
Inventors: |
Evans; Christopher J. (Venice,
CA), Keith, Jr.; Duane E. (Woodland Hills, CA), Edwards;
Robert H. (Los Angeles, CA) |
Assignee: |
The Regents of the University of
California (Oakland, CA)
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Family
ID: |
25457474 |
Appl.
No.: |
08/411,859 |
Filed: |
March 28, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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929200 |
Aug 13, 1992 |
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Current U.S.
Class: |
435/69.1;
435/252.3; 435/254.2; 435/320.1; 536/23.5; 435/365; 435/325;
435/348 |
Current CPC
Class: |
C07K
14/705 (20130101); A61P 25/04 (20180101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/705 (20060101); C12N
015/12 (); C07K 014/705 () |
Field of
Search: |
;435/69.1,240.2,252.3,254.11,320.1,325,365,254.2,348 ;536/23.5 |
Other References
Libert et al., Science, 244, 569-572, 1989. .
Gramsch et al., "Monoclonal Anti-idiotypic Antibodies to Opioid
Receptors," Jour of Biological Chem, vol. 263, No. 12, pp.
5853-5859 (1988). .
Machida et al., "Three Technical Approaches for Cloning Opioid
Receptors," Natl Institute on Drug Abuse Research Monograph Series,
pp. 93-110, (1988). .
Simonds et al., "Purification of the opiate receptor of NG108-15
neuroblastoma-glioma hybrid cells," Proc. Natl. Acad. Sci.,vol. 82,
pp. 4974-4978 (1985). .
Smith et al., "Problems and Approaches in Studying Membrane Opioid
Receptors," Natl Institute on Drug Abuse Research Monograph Series,
pp. 69-84, (1991). .
Eberwine et al (1987) Fed. Proc. 46(4): 1444 (Abstr.#6582). .
Evans et al (1992a) Soc. Neurosci: Abstr. 18(1): 21 (Abstr. #16.1).
.
Evans et al (1992b) Science 258: 1952-1955. .
Xie et al (1992) Proc. Nat'l Acad Sci. 89: 4124-4128. .
Yu et al (1986) J. Biol. Chem. 261(3): 1065-1090. .
Carr et al (1989) Immunol. Lett. 20: 181-186. .
Sanger et al (1977) Proc. Nat'Acad Sci. 74(12): 5463-5467. .
Davis et al (1986) "Basic Methods in Molecular Biology", Elsevier
Science Publishing Co., Inc. New York., pp. 1-372. (Table of
Contents provided). .
Kieffer et al (1992) Proc. Nat'l Acad Sci. 89: 12048-12052. .
Bochet et al., Molecular Pharmacology (1988) 34: 436-443. .
Law et al., Molecular Pharmacology (1982) 21 :438-91. .
Schofield et al., The EMBO Journal (1989) 8(2):489-495. .
Xie et al., Proc. Natl. Acad. Sci. (1992) 89:4124-4128..
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Primary Examiner: Teng; Sally P.
Attorney, Agent or Firm: MorrisonFoersterLLP
Government Interests
This invention was made with Government support under Grant No.
DA05010 awarded by the Alcohol, Drug Abuse and Mental Health
Administration. The Government has certain rights in the invention.
Parent Case Text
This application is a continuation of application Ser. No.
07/929,200, filed Aug. 13, 1992, now abandoned.
Claims
We claim:
1. An isolated and purified nucleic acid molecule which comprises a
nucleotide sequence encoding a mammalian delta opioid receptor,
said nucleotide sequence selected from the group consisting of:
a) a nucleotide sequence encoding the amino acid sequence of SEQ ID
NO: 2;
b) a nucleotide sequence encoding the amino acid sequence of SEQ ID
NO: 10; and
c) a nucleotide sequence encoding the amino acid sequence encoded
by a nucleotide sequence that hybridizes under conditions of low
stringency with the complement of SEQ ID NO: 1.
2. A DNA molecule comprising the nucleotide sequence according to
claim 1, wherein said nucleotide sequence is operably linked to a
promoter.
3. A host cell comprising the DNA molecule of claim 2.
4. The host cell of claim 3 which is selected from the group
consisting of yeast cells, mammalian cells, insect cells and
bacteria.
5. The host cell of claim 4, which is a COS-7 cell.
6. A method of producing host cells that express a mammalian delta
opioid receptor, which method comprises the steps of obtaining the
DNA molecule of claim 2, transforming said DNA molecule into host
cells, and isolating host cells that express the mammalian delta
opioid receptor.
7. A host cell prepared by the method of claim 6.
8. The DNA molecule of claim 2 wherein said nucleotide sequence
encodes the delta opioid receptor of SEQ ID NO: 2.
9. The nucleic acid molecule of claim 1 wherein the receptor is the
human delta opioid receptor.
10. The nucleic acid molecule of claim 1 wherein said nucleotide
sequence encodes the delta opioid receptor of SEQ ID NO: 2.
11. A method of obtaining an isolated DNA molecule which encodes a
delta opioid receptor of a particular mammal, comprising the steps
of constructing a genomic or cDNA library prepared from the mammal,
probing said library with a probe comprising the nucleotide
sequence of SEQ ID NO: 1 under low stringency conditions, isolating
DNA molecules to which said probe hybridizes.
12. A nucleic acid molecule comprising a nucleotide sequence which
is the complement of the nucleotide sequence of SEQ ID NO: 1.
Description
TECHNICAL FIELD
The invention relates to substances involved in vertebrate nervous
systems, and in particular to the delta opioid receptor and
activities mediated thereby. Accordingly, the invention concerns
recombinant materials useful for the production of delta opioid
receptor, the receptor as a diagnostic tool, therapeutic and
diagnostic compositions relevant to the receptor, and methods of
using the receptor to screen for drugs that modulate the activity
of the receptor.
BACKGROUND ART
The term "opioid" generically refers to all drugs, natural and
synthetic, that have morphine-like actions. Formerly, the term
"opiate" was used to designate drugs derived from opium, e.g.,
morphine, codeine, and many semi-synthetic congeners of morphine.
After the isolation of peptide compounds with morphine-like
actions, the term opioid was introduced to refer generically to all
drugs with morphine-like actions. Included among opioids are
various peptides that exhibit morphine-like activity, such as
endorphins, enkephalins and dynorphins. However, some sources have
continued to use the term "opiate" in a generic sense, and in such
contexts, opiate and opioid are interchangeable. Additionally, the
term opioid has been used to refer to antagonists of morphine-like
drugs as well as to characterize receptors or binding sites that
combine with such agents.
Opioids are generally employed as analgesics, but they may have
many other pharmacological effects as well. Morphine and related
opioids produce their major effects on the central nervous and
digestive systems. The effects are diverse, including analgesia,
drowsiness, mood changes, respiratory depression, dizziness, mental
clouding, dysphoria, pruritus, increased pressure in the biliary
tract, decreased gastrointestinal motility, nausea, vomiting, and
alterations of the endocrine and autonomic nervous systems.
A significant feature of the analgesia produced by opioids is that
it occurs without loss of consciousness. When therapeutic doses of
morphine are given to patients with pain, they report that the pain
is less intense, less discomforting, or entirely gone. In addition
to experiencing relief of distress, some patients experience
euphoria. However, when morphine in a selected pain-relieving dose
is given to a pain-free individual, the experience is not always
pleasant; nausea is common, and vomiting may also occur.
Drowsiness, inability to concentrate, difficulty in mentation,
apathy, lessened physical activity, reduced visual acuity, and
lethargy may ensue.
The development of tolerance and physical dependence with repeated
use is a characteristic feature of all opioid drugs, and the
possibility of developing psychological dependence on the effect of
these drugs is a major limitation for their clinical use. There is
evidence that phosphorylation may be associated with tolerance in
selected cell populations. (Louie, A. et al. Biochem. Biophys. Res.
Comm., 152: 1369-75 (1988)).
Acute opioid poisoning may result from clinical overdosage,
accidental overdosage, or attempted suicide. In a clinical setting,
the triad of coma, pinpoint pupils, and depressed respiration
suggest opioid poisoning. Mixed poisonings including agents such as
barbiturates or alcohol may also contribute to the clinical picture
of acute opioid poisoning. In any scenario of opioid poisoning,
treatment must be administered promptly.
The opioids interact with what appear to be several closely related
receptors. Various inferences have been drawn from data that have
attempted to correlate pharmacologic effects with the interactions
of opioids with a particular constellation of opioid receptors.
(Goodman and Gilman's, The Pharmacological Basis of Therapeutics,
7th ed 493-95 (MacMillan 1985)). For example, analgesia has been
associated with mu and kappa receptors. Delta receptors are
believed to be involved in alterations of affective behavior; this
belief is based primarily on the localization of these receptors in
limbic regions of the brain. Additionally, activation, e.g., ligand
binding with stimulation of further receptor-mediated response, of
delta opioid receptors is believed to inhibit the release of other
neurotransmitters. The paths containing relatively high populations
of delta opioid receptor are similar to the paths implicated to be
involved in Huntington's disease. Accordingly, it is postulated
that Huntington's disease may correlate with some effect on delta
opioid receptors.
Pharmacologically, it has been found that there are two distinct
classes of opioid molecules that can bind opioid receptors: the
opioid peptides (e.g., the enkephalins, dynorphins, and endorphins)
and the alkaloid opiates (e.g., morphine, etorphine, diprenorphine
and naloxone). Subsequent to the initial demonstration of opiate
binding sites (Pert, C. B. and Snyder, S. H., Science 179:1011-1014
(1973)), the differential pharmacological and physiological effects
of both opioid peptide analogues and alkaloid opiates served to
delineate multiple opioid receptors. Accordingly, three
anatomically and pharmacologically distinct opioid receptor types
have been described: delta, kappa and mu. Furthermore, each type is
believed to have subtypes. (Wollemann, M., J. Neurochem.,
54(4):1095-1101 (1990); Lord, J. A., et al., Nature, 267:495-499,
(1977)).
All three of these opioid receptor types appear to share the same
functional mechanisms at a cellular level. For example, the opioid
receptors cause inhibition of adenylate cyclase, and inhibition of
neurotransmitter release via both potassium channel activation and
inhibition of Ca.sup.2+ channels (Evans, C. J., In: Biological
Basis of Substance Abuse, S. G. Korenman & J. D. Barchas, eds.,
Oxford University Press (in press); North, A. R., et al., Proc.
Natl. Acad. Sci. USA, 87(18): 7025-29 (1990); Gross, R. A., et al.,
Proc. Natl. Acad. Sci. U S A, 87(18): 7025-29 (1990); Sharma, S.
K., et al., Proc. Natl. Acad. Sci. U.S.A., 72:(8) 3092-96 (1975)).
Although the functional mechanisms are the same, the behavioral
manifestations of receptor-selective drugs differ greatly.
(Gilbert, P. E. & Martin, W. R., J. Pharmacol. Exp. Ther.,
198(1):66-82 (1976)). Such differences may be attributable in-part
to the anatomical location of the different receptors.
Delta opioid receptors are of particular relevance for the present
invention. Delta receptors have a more discrete distribution within
the mammalian CNS than either mu or kappa receptors, with high
concentrations in the amygdaloid complex, striatum, substantia
nigra, olfactory bulb, olfactory tubercles, hippocampal formation,
and the cerebral cortex. (Mansour, A., et al., Trends in Neurosci.,
11(7): 308-14 (1988)). The rat cerebellum is remarkably devoid of
opioid receptors including delta opioid receptors.
Several opioid molecules are known to selectively or preferentially
bind delta receptors. Of the vertebrate endogenous opioids, the
enkephalins, particularly met-enkephalin and leu-enkephalin, appear
to possess the highest affinity for delta receptors, although the
enkephalins also have high affinity for mu receptors. Additionally,
the deltorphans, peptides isolated from frog skin, comprise a
family of opioid peptides that have high affinity and selectivity
for delta receptors. (Erspamer, V., et al., Proc. Natl. Acad. Sci.
U S A, 86(13): 5188-92 (1989)).
A number of synthetic enkephalin analogues are also delta-selective
including:
(D-Ser.sup.2) leucine enkephalin Thr (DSLET) (Garcel, G., et al.,
F.E.B.S. Letters 118(2): 245-247 (1980)); and
(D-Pen.sup.2, D-Pen.sup.5) enkephalin (DPDPE) (Akiyama, K., et al.,
P.N.A.S. 82: 2543-2547 (1985))
Recently a number of other selective delta ligands have been
synthesized, and their bioactivities and binding characteristics
suggest the existence of more than one delta receptor subtype.
(Takemori, A. E., et al., Annual Review of Pharmacology and
Toxicology, 32:239-69 (1992); Negri, L., et al., Eur. J.
Pharmacol., 196:355-335 (1991); Sofuoglu, M., et al.,
Pharmacologist 32:151 (1990)).
The synthetic pentapeptide 2dAla, 5dLeu enkephalin (DADLE) was
considered to be delta-selective; although DADLE shows high
affinity for delta receptors, it also binds equally well to mu
receptors. The synthetic peptide D-Ala.sup.2, N-Me-Phe.sup.4,
Gly-ol.sup.5 -enkephalin (DAGO) has been found to be a selective
ligand for mu-receptors.
The existence of multiple delta opioid receptors has been implied
not only from the pharmacological studies addressed above, but also
from molecular weight estimates obtained by use of irreversible
affinity ligands. These studies indicate molecular weights for the
delta opioid receptor that range from 30,000-60,000 daltons.
(Evans, C. J., In: Biological Basis of Substance Abuse, S. G.
Korenman & J. D. Barchas, Eds., Oxford University Press (in
press); Bochet, P., et al., Mol. Pharmacol., 34(4):436-43 (1988)).
The various receptor sizes may represent alternative splice
products, although this has not been established.
Many studies of the delta opioid receptor have been performed with
the neuroblastoma/glioma cell line NG108-15. The NG108-15 cell line
was generated by fusion of the rat glial cell line (C6BU-1) and the
mouse neuroblastoma cell line (N18-TG2) (Klee, W. A. and Nirenberg,
M. A., P.N.A.S. USA 71(9): 3474-3477 (1974)). The rat glial cell
line expresses essentially no delta opioid receptors, whereas the
mouse neuroblastoma cell line expresses low amounts of the
receptor. Thus, a mouse chromosomal origin of the delta opioid
receptors in the NG108-15 cells has been suggested. (Law, Mol.
Pharm., 21: 438-91).
Each NG108-15 cell is estimated to express approximately 300,000
delta-receptors. Only delta-type opioid receptors are expressed,
although it is not known whether these represent more than a single
subtype.
Thus, the NG108-15 cell line has served to provide considerable
insight into the binding characterization of opioid receptors,
particularly delta opioid receptors. However, the NG108-15 cell
line is a cancer-hybrid, and it may not be completely
representative of the delta receptor in endogenous neurons due to
the unique cellular environment in the hybrid cells.
An extensive literature has argued that the opioid receptors are
coupled to G-proteins (see, e.g., Schofield, P. R., et al., Embo
J., 8(2):489-95 (1989)), and are thus members of the family of
G-protein coupled receptors. G-proteins are guanine nucleotide
binding proteins that couple the extracellular signals received by
cell surface receptors to various intracellular second messenger
systems. Identified members of the G-protein-coupled family share a
number of structural features, the most highly conserved being
seven apparent membrane-spanning regions, which are highly
homologous among the members of this family. (Strosberg, A. D.,
Eur. J. Biochem. 196(l):1-10 (1991)). Evidence that the opioid
receptors are members of this family includes the stimulation of
GTPase activity by opioids, the observation that GTP analogues
dramatically effect opioid and opiate agonist binding, and the
observation that pertussis toxin (which by ADP ribosylation
selectively inactivates both the Gi and Go subfamilies of
G-proteins) blocks opioid receptor coupling to adenylate cyclase
and to K.sup.+ and Ca.sup.2+ channels. (Evans, C. J., In:
Biological Basis of Substance Abuse, S. G. Korenman & J. D.
Barchas, Eds., Oxford University Press (in press)).
The members of the G-protein-coupled receptor family exhibit a
range of characteristics. Many of the G-protein-coupled receptors,
e.g., the somatostatin receptor and the angiotensin receptor, have
a single exon that encodes the entire protein coding region
(Strosberg, A. D., Eur. J. Biochem. 196(1):1-10 (1991); Langord,
K., et al., B.B.R.C. 138(3): 1025-1032 (1992)). However, others,
such as substance-P receptor and the Dopamine D2 receptor contain
the protein coding region. The D2 receptor is particularly
interesting in that alternate splicing of the message gene gives
rise to different transcribed products (i.e., receptors). (Evans,
C. J., In: Biological Basis of Substance Abuse, S. G. Korenman
& J. D. Barchas, Eds., Oxford University Press (in press);
Strosberg, A. D., Eur. J. Biochem. 196(1):1-10 (1991)).
Interestingly, somatostatin ligands are reported to bind to opioid
receptors (Terenius, L., Eur. J. Pharmacol. 38: 211 (1976); Mulder,
A. H., et al., Eur. J. Pharmacol. 205:1-6 (1991)) and, furthermore,
to have similar molecular mechanisms. (Tsunoo, A., et al., P.N.A.S.
83: 9832-9836 (1986)).
In previous efforts to describe and purify opioid receptors two
clones have been described that were hypothesized either to encode
opioid receptors or a portion thereof. The first clone, which
encodes opiate binding protein OBCAM (Schofield, P. R., et al.,
Embo J., 8(2):489-95 (1989)) was obtained by utilizing a probe
designed from an amino acid sequence contained in protein purified
on a morphine affinity column. OBCAM does not have membrane
spanning domains; however, it has a C-terminal domain that is
characteristic of attachment of the protein to the membrane by a
phosphatidylinositol linkage. This feature, which is shared by
members of the immunoglobulin superfamily, is not common to the
family of receptors coupled to G-proteins. Thus, it has been
proposed that OBCAM is part of a receptor complex along with other
components that are coupled to G-proteins. (Schofield, P. R., et
al., Embo J., 8(2):489-95 (1989)). At present, however, there is no
direct evidence for such a complex.
A second proposed opioid receptor clone was obtained in an effort
to clone a receptor that binds kappa opioid receptor ligands. (Xie,
G. X., Proc. Natl. Acad. Sci. USA, 89: 4124-4128 (1992)). A DNA
encoding a G-coupled receptor from a placental cDNA library was
isolated. This receptor has an extremely high homology with the
neurokinin B receptor (84% identical throughout the proposed
protein sequence). When this clone was expressed in COS cells, it
displayed opioid peptide displaceable binding of .sup.3
H-Bremazocine (an opiate ligand with high affinity for kappa
receptors). However, the low affinity of this receptor for .sup.3
H-Bremazocine, and the lack of appropriate selectivity since this
receptor binds both mu and delta ligands, makes it doubtful that
this cloned molecule is actually an opioid receptor. Furthermore,
characterization of opioid receptor proteins has proven difficult
because of the instability of these membrane-bound receptors after
they are solubilized, and purified delta opioid receptors have not
been isolated. The previous reports estimating the molecular
weights for opioid receptor proteins in the wide range from
30,000-60,000 daltons reflect the difficulty in isolating and
characterizing this protein.
DISCLOSURE OF THE INVENTION
The invention provides recombinant materials and methods to produce
mammalian delta opioid receptor. Methods for isolating the
receptor, isolating the gene that encodes the receptor,
recombinantly producing the receptor, and methods for using the
receptor to screen for drugs that modulate the activity of the
receptor are also provided.
Thus, in one aspect, the invention is directed to recombinant
materials and methods for the production of a mammalian delta
opioid receptor protein. Such materials and methods include
isolated and purified forms or recombinantly produced forms of DNA
encoding said delta opioid receptor protein, expression systems
suitable for the production of the protein, and cells transformed
with said expression systems. Especially useful are mammalian cells
which express the gene in such a way that the delta opioid receptor
protein is displayed at the surface of the cells. The cells of this
type are especially useful products of the invention, since they
offer means to screen native and synthetic candidate agonists and
antagonists from the ligands which bind delta opioid receptors.
In still other aspects, the invention is directed to methods to
screen candidate agonists and/or antagonists for ligands that
activate the delta opioid receptors using the recombinant
transformed cells of the invention. Such assays include binding
assays using competition with ligands known to bind delta opioid
receptors; agonist assays which analyze the transformed cells for
activation of the secondary pathways associated with opioid
receptor activation; and assays which evaluate the effect on
binding of the candidate to the receptor by the presence or absence
of sodium ion and GTP. Antagonist assays include the combination of
the ability of the candidate to bind the receptor while failing to
effect further activation, and, more importantly, competition with
a known agonist. Still another aspect of the invention is provision
of antibody compositions which are immunoreactive with the delta
opioid receptor protein. Such antibodies are useful, for example,
in purification of the receptors after solubilization or after
recombinant production thereof.
In still other aspects, the invention is directed to probes useful
for the identification of DNA which encodes related opioid
receptors in various species or of different types and
subtypes.
Accordingly, an object of the present invention is to provide an
isolated and purified form of a DNA sequence encoding a delta
opioid receptor.
Another object is to provide a recombinantly produced DNA sequence
encoding a delta opioid receptor.
Another object is to produce an antisense sequence corresponding to
known sense sequence encoding the delta opioid receptor.
Another object of the invention is to provide a DNA construct
comprised of a control sequence operatively linked to a DNA
sequence which encodes a delta opioid receptor and to provide
recombinant host cells transformed with the DNA construct.
Another object is to isolate, clone and characterize, from various
vertebrate species, DNA sequences encoding the various related
receptors, by hybridization of the DNA derived from such species
with a native DNA sequence encoding the delta opioid receptors of
the invention.
An advantage of the present invention is that delta opioid receptor
encoding DNA sequences can be expressed at the surface of host
cells which can conveniently be used to screen drugs for their
ability to interact with and/or bind to the receptors.
These and other objects, advantages and features of the present
invention will become apparent to those persons skilled in the
art.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts a comparison between .sup.3 H-Diprenorphine
saturation curve of NG108-15 cells and COS cells three days
following electroporation with DOR-1 in the CDM8 vector. Specific
opioid binding was undetectable in nontransfected COS cells or COS
cells transfected with plasmid alone.
FIG. 2 depicts displacement curves of 5 nM 3H-Diprenorphine from
COS cell membranes of cells transfected with DOR-1. The
3H-Diprenorphine was displaced by diprenorphine, etorphine,
morphine and levorphanol, but not by dextrorphan, the non-opiate
active optical isomer of levorphanol.
FIG. 3 depicts displacement curves of 5 nm 3H-Diprenorphine from
COS cell membranes of cells transfected with DOR-1. The
3H-Diprenorphine was displaced by DPDPE and DSLET, delta-selective
agonists; by DADLE, a high affinity ligand for mu and delta
receptors; and by Dynorphin 1-17, a kappa-preferring ligand. It was
not displaced by DAGO, a mu-selective ligand.
FIG. 4 depicts the results of a Northern Blot of mRNA from NG108-15
cells and cells from various rat brain regions.
FIG. 5 is the DNA sequence encoding the DOR-1 delta opioid
receptor, and the corresponding amino acid sequence of said delta
opioid receptor (SEQ ID NO: 1 and SEQ ID NO: 2).
FIG. 6 depicts the deduced protein sequence of DOR-1, compared with
the rat somatostatin receptor (SEQ ID NO: 2 and SEQ ID NO: 3).
Consensus glycosylation sites predicted to fall in extracellular
domains are indicated by an asterisk. Potential protein kinase C
sites are listed in example 5. The seven predicted membrane
spanning regions are underlined. These seven regions are predicted
based on the hydrophobicity profile and published predictions
(MacVector software program (IBI); T. Hopp, and K. Woods, Proc.
Natl. Acad. Sci. USA 78, 3842-3828 (1981)). For sequencing, the
cDNA insert was subcloned into pBluescript (Strategene), and both
strands were sequenced from single-stranded DNA using Sequenase and
Taq cycle sequencing (USB). For ambiguities due to compressions
7-deaza-dGTP replaced dGTP in the sequencing reactions and the
products were resolved on formamide gels.
FIG. 7 depicts a Southern blot of radiolabeled DOR-1 cDNA probe
hybridized at high stringency to NG108-15, mouse, rat and human DNA
cut with BamHI.
MODES OF CARRYING OUT THE INVENTION
The invention provides DNA encoding mammalian delta opioid receptor
protein and additional recombinant materials and methods useful for
the production of this protein. In addition, eucaryotic cells, such
as COS cells, transformed with the recombinant materials of the
invention so as to express delta opioid receptor protein at their
surface are useful in screening assays to identify candidate opioid
agonists and antagonists. In addition, antibodies may be raised to
the recombinantly produced delta opioid receptor protein. These
antibodies are useful in immunoassays for said protein and in
affinity purification thereof.
Recombinant Delta Opioid Receptor
Illustrated hereinbelow is the obtention of a cDNA encoding murine
delta opioid receptor. The complete DNA sequence of the cDNA, and
the amino acid sequence encoded thereby are set forth herein in
FIG. 5. The availability of this cDNA permits the retrieval of the
corresponding delta opioid receptors-encoding DNA from other
vertebrate species. Accordingly, the present invention places
within the possession of the art, recombinant materials and methods
for the production of cells expressing delta opioid receptors of
various subtypes and of various vertebrate species. Thus, the cDNA
of FIG. 5, or a portion thereof, may be used as a probe to identify
that portion of vertebrate genomic DNA which encodes the
corresponding delta opioid receptor protein. Sample methods as used
to prepare the relevant genomic library and identify the delta
opioid receptor-encoding gene are described for convenience
hereinbelow.
In the alternative, the DNA of FIG. 5 or a portion thereof may be
used to identify specific tissues or cells which express delta
opioid receptor protein by analyzing the messenger RNA, for
example, using Northern blot techniques. Those tissues which are
identified as containing mRNA encoding delta opioid receptor
protein using the probes of the invention are then suitable sources
for preparation of cDNA libraries which may further be probed using
the cDNA described hereinbelow.
The DNA encoding the various vertebrate delta opioid receptor
proteins, obtained in general as set forth above, according to the
standard techniques described hereinbelow, can be used to produce
cells which express the delta opioid receptor at their surface;
such cells are typically eucaryotic cells, in particular, mammalian
cells such as COS cells or CHO cells. Suitable expression systems
in eucaryotic cells for such production are described hereinbelow.
The delta opioid receptor proteins may also be produced in
procaryotes or in alternative eucaryotic expression systems for
production of the protein per se. The protein may be ligated into
expression vectors preceded by signal sequences to effect its
secretion, or may be produced intracellularly, as well as at the
cell surface, depending on the choice of expression system and
host. If desired, the delta opioid receptor protein thus
recombinantly produced may be purified using suitable means of
protein purification, and, in particular, antibodies or fragments
thereof immunospecific for the delta opioid receptor protein.
Screening for Opioid Agonists and Antagonists Using Recombinant
Cells
The ability of a candidate compound to behave as an opioid agonist
or antagonist may be assessed using the recombinant cells of the
invention in a variety of ways. To exhibit either agonist or
antagonist activity, the candidate compound must bind to the opioid
receptor. Thus, to assess the ability of the candidate to bind,
either a direct or indirect binding assay may be used. For a direct
binding assay, the candidate binding compound is itself labeled,
such as with a radioisotope or fluorescent label, and binding to
the recombinant cells of the invention is assessed by comparing the
acquisition of label by the recombinant cells to the acquisition of
label by untransformed corresponding cells.
More convenient, however, is the use of a competitive assay wherein
the candidate compound competes for binding to the recombinant
cells of the invention with a labeled form of an opioid ligand
known to bind to the receptor. Such ligands are themselves labeled
using radioisotopes or fluorescent moieties, for example. A
particularly suitable opioid known to bind to this receptor is
diprenorphine. A typical protocol for such an assay is as
follows:
In general, about 10.sup.6 recombinant cells are incubated in
suspension in 1.0 ml of Kreb's Ringer Hepes Buffer (KRHB) at pH
7.4, 37.degree. C. for 20 min with .sup.3 H-diprenorphine.
Nonspecific binding is determined by the addition of 400 nM
diprenorphine in the binding mixtures. Various concentrations of
candidate compounds are added to the reaction mixtures. The
incubations are terminated by collecting the cells on Whatman GF-B
filters, with removal of excess radioactivity by washing the
filters three times with 5 ml of KRHB at 0.degree. C. After
incubating at 20.degree. C. overnight in 5 ml of Liquiscint
(National Diagnostics, Somerville, N.J.), the radioactivity on the
filters is determined by liquid scintillation counting.
The K.sub.d (dissociation constant) values for the candidate opiate
ligands can be determined from the IC.sub.50 value. (i.e., the
concentration of ligand that results in a 50% decrease in binding
of labeled diprenorphine).
The effects of sodium and GTP on the binding of ligands to the
recombinantly expressed receptors can be used to distinguish
agonist from antagonist activities. If the binding of a candidate
compound is sensitive to Na.sup.+ and GTP, it is more likely to be
an agonist than an antagonist, since the functional coupling of
delta opioid receptors to second messenger molecules such as
adenylate cyclase requires the presence of both sodium and GTP.
(Blume, et al., P.N.A.S. U.S.A., 23: 26-35 (1979)). Furthermore,
sodium, GTP, and GTP analogues have been shown to effect the
binding of opioids and opioid agonists to opioid receptors. (Blume,
Life Sciences, 22: 1843-52 (1978)). Since opioid antagonists do not
exhibit binding that is sensitive to guanine nucleotides and
sodium, this effect is used as a method for distinguishing agonists
from antagonists using binding assays.
In addition, agonist activity can directly be assessed by the
functional result within the cell. For example, it is known that
the binding of opioid agonists inhibits cAMP formation, inhibits
potassium channel activation, inhibits calcium channel activation,
and stimulates GTPase. Assessment of these activities in response
to a candidate compound is diagnostic of agonist activity. In
addition, the ability of a compound to interfere with the
activating activity of a known agonist such as etorphine
effectively classifies it as an antagonist.
In one typical assay, the measurement of cAMP levels in cells
expressing delta opioid receptors is carried out by determining the
amount of .sup.3 H-cyclic AMP (cAMP) formed from intracellular ATP
pools prelabeled with .sup.3 H-adenine. (Law, et al., Mol.
Pharmacol., 21:483-91 (1982)). Thus, cAMP formation assays are
carried out with 0.5.times.10.sup.6 cells/0.5 ml of KRHB.sup.2 at
pH 7.4, incubated at 37.degree. C. for 20 minutes. After addition
of the internal standard .sup.32 P-cyclic AMP, the radioactive
cyclic AMP is separated from other .sup.3 H-labeled nucleotides by
known double-column chromatographic methods. The opiate agonists'
ability to inhibit cyclic AMP accumulation is then determined as
described by Law, et al., Mol. Pharmacol., 21:483-91 (1982).
The potency of a candidate opiate antagonist can be determined by
measuring the ability of etorphine to inhibit cyclic AMP
accumulation in the presence and in the absence of known amounts of
these candidate antagonists. The inhibition constant (K.sub.i) of
an antagonist can then be calculated from the equation for
competitive inhibitors.
An interesting feature of screening assays using the prior art
NG108-15 cells is that the agonist adenylate cyclase inhibition
function apparently does not require binding of all receptors on
these cells. Thus, the inhibition constant and the dissociation
constant for the opioid ligands differed for these cells.
The foregoing assays, as described above, performed on the
recombinantly transformed cells of the invention, form a more
direct and more convenient screen for candidate compounds having
agonist and antagonist delta opioid receptor activity than that
previously available in the art. Furthermore, such assays are more
sensitive since cells can, in accordance with the present
invention, be engineered to express high levels of the delta opioid
receptor. Additionally, cells engineered in accordance with the
present invention will circumvent the concern attendant to NG108-15
cells, that the cellular environment of that cancer hybrid
artifactually effects the delta opioid receptor expressed
thereon.
Methods to Prepare Delta Opioid Receptor Protein or Portions
Thereof
The present invention provides the amino acid sequence of a murine
delta opioid receptor; similarly, the availability of the cDNA of
the invention places within possession of the art corresponding
vertebrate opioid receptors whose amino acid sequence may also be
determined by standard methods. As the amino acid sequences of such
opioid receptors are known, or determinable, in addition to
purification of such receptor protein from native sources,
recombinant production or synthetic peptide methodology may also be
employed.
The delta opioid receptor or portions thereof can thus also be
prepared using standard solid phase (or solution phase) peptide
synthesis methods, as is known in the art. In addition, the DNA
encoding these peptides may be synthesized using commercially
available oligonucleotide synthesis instrumentation for production
of the protein in the manner set forth above. Production using
solid phase peptide synthesis is, of course, required if
non-gene-encoded amino acids are to be included.
The nomenclature used to describe the peptides and proteins of the
invention follows the conventional practice where the N-terminal
amino group is assumed to be to the left and the carboxy group to
the right of each amino acid residue in the peptide. In the
formulas representing selected specific embodiments of the present
invention, the amino- and carboxy-terminal groups, although often
not specifically shown, will be understood to be in the form they
would assume at physiological pH values, unless otherwise
specified. Thus, the N-terminal NH3.sup.+ and C-terminal COO.sup.-
at physiological pH are understood to be present though not
necessarily specified and shown, either in specific examples or in
generic formulas. Free functional groups on the side chains of the
amino acid residues may also be modified by glycosylation,
phosphorylation, cysteine binding, amidation, acylation or other
substitution, which can, for example, alter the physiological,
biochemical, or biological properties of the compounds without
affecting their activity within the meaning of the appended
claims.
In the peptides shown, each gene-encoded residue, where
appropriate, is represented by a single letter designation,
corresponding to the trivial name of the amino acid, in accordance
with the following conventional list:
______________________________________ One-Letter Three-letter
Amino Acid Symbol Symbol ______________________________________
Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic acid D Asp
Cysteine C Cys Glutamine Q Gln Glutamic acid E Glu Glycine G Gly
Histidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys
Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Ser
Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val
______________________________________
Nomenclature of Enkephalins
Enkephalins are either of two peptides of 5 residues with the
N-terminal residue numbered 1:
tyr-gly-gly-phe-xxx (SEQ ID NO:4) 1 2 3 4 5
In "met enkephalin" the 5th residue is methionine:
In "leu enkephalin" the 5th residue is leucine:
Analogs can be made with (i) amino acid substitutions, (ii) D-amino
acid substitutions, and/or (iii) additional amino acids. The site
at which the substitution is made is noted at the beginning of the
compound name. For example, "(D-ala.sup.2, D-leu.sup.5) enkephalin"
means that D-ala is present at the second position and D-leu is
present at the fifth position:
One letter abbreviations can also be used. Thus, "(D-ser.sup.2) leu
enkephalin" could be abbreviated as "DSLE." Additional residues are
noted as well. Thus, the addition of a threonine residue (to the
sixth position) of (D-ser.sup.2) leu enkephalin would be
"(D-ser.sup.2) leu enkephalin thr" which could be abbreviated as
"DSLET":
Antibodies
Antibodies immunoreactive with critical positions of the delta
opioid receptor can be obtained by immunization of suitable
mammalian subjects with peptides, containing as antigenic regions
those portions of the receptor intended to be targeted by the
antibodies. Certain protein sequences have been determined to have
a high antigenic potential. Such sequences are listed in antigenic
indices, for example, MacVector software (I.B.I.). Thus, by
determining the sequence of the delta opioid receptor protein then
evaluating the sequence with an antigenic index probable antigenic
sequences are located.
Antibodies are prepared by immunizing suitable mammalian hosts
according to known immunization protocols using the peptide haptens
alone, if they are of sufficient length, or, if desired, or if
required to enhance immunogenicity, conjugated to suitable
carriers. Methods for preparing immunogenic conjugates with
carriers such as BSA, KLH, or other carrier proteins are well known
in the art. In some circumstances, direct conjugation using, for
example, carbodiimide reagents may be effective; in other instances
linking reagents such as those supplied by Pierce Chemical Co.,
Rockford, Ill., may be desirable to provide accessibility to the
hapten. The hapten peptides can be extended or interspersed with
cysteine residues, for example, to facilitate linking to carrier.
Administration of the immunogens is conducted generally by
injection over a suitable time period and with use of suitable
adjuvants, as is generally understood in the art. During the
immunization schedule, titers of antibodies are taken to determine
adequacy of antibody formation.
While the polyclonal antisera produced in this way may be
satisfactory for some applications, for pharmaceutical
compositions, use of monoclonal preparations is preferred.
Immortalized cell lines which secrete the desired monoclonal
antibodies may be prepared using the standard method of Kohler and
Milstein or modifications which effect immortalization of
lymphocytes or spleen cells, as is generally known. The
immortalized cell lines secreting the desired antibodies are
screened by immunoassay in which the antigen is the peptide hapten
or is the delta opioid receptor itself displayed on a recombinant
host cell. When the appropriate immortalized cell culture secreting
the desired antibody is identified, the cells can be cultured
either in vitro or by production in ascites fluid.
The desired monoclonal antibodies are then recovered from the
culture supernatant or from the ascites supernatant. Fragments of
the monoclonals or the polyclonal antisera which contain the
immunologically significant portion can be used as antagonists, as
well as the intact antibodies. Use of immunologically reactive
fragments, such as the Fab, Fab', of F(ab').sub.2 fragments is
often preferable, especially in a therapeutic context, as these
fragments are generally less immunogenic than the whole
immunoglobulin.
Standard Methods
The techniques for sequencing, cloning and expressing DNA sequences
encoding the amino acid sequences corresponding to a delta opioid
receptor, e.g. polymerase chain reaction (PCR), synthesis of
oligonucleotides, probing a cDNA library, transforming cells,
constructing vectors, preparing antisense oligonucletide sequences
based on known sense nucleotide sequences, extracting messenger
RNA, preparing cDNA libraries, and the like are well-established in
the art. Skilled artisans are familiar with the standard resource
materials for specific conditions and procedures. The following
paragraphs are provided for convenience, it being understood that
the invention is limited only by the appended claims.
RNA Preparation and Northern Blot
RNA preparation is as follows: The samples used for preparation of
RNA are immediately frozen in liquid nitrogen and then stored until
use at -80.degree. C. The RNA is prepared by CsCl centrifugation
(Ausubel et al., supra) using a modified homogenization buffer
(Chirgwin et al., Biochem. 18:5294-5299 (1979)). Poly (A.sup.+)RNA
is selected by oligo(dT) chromatography (Aviv and Leder, Proc.
Natl. Acad. Sci. USA 69:1408-1412 (1972)). RNA samples are stored
at -80.degree. C.
Analysis of gene expression and tissue distribution can be
accomplished using Northern blots using, e.g., radiolabeled probes.
The mRNA is size separated using gel electrophoresis and then
typically is transferred to a nylon membrane or to nitrocellulose,
and hybridized with radiolabeled probe. Presence of the hybridized
probe is detected using autoradiography.
Cloning
The cDNA sequences encoding the delta opioid protein were obtained
from a random-primed, size-selected cDNA library.
Alternatively, the cDNA sequences encoding delta opioid receptor
protein are obtained from a cDNA library prepared from mRNA
isolated from cells expressing the receptor protein in various
organs such as the brain, according to procedures described in
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, second
edition, Sambrook, et al., eds. (1989).
The cDNA insert from the successful clone, excised with a
restriction enzyme such as EcoRI, is then used as a probe of the
original cDNA library or other libraries (low stringency) to obtain
the additional clones containing inserts encoding other regions of
the protein that together or alone span the entire sequence of
nucleotides coding for the protein.
An additional procedure for obtaining cDNA sequences encoding the
delta opioid receptor protein is PCR. PCR is used to amplify
sequences from a pooled cDNA library of reversed-transcribed RNA,
using oligonucleotide primers based on the transporter sequences
already known.
Vector Construction
Construction of suitable vectors containing the desired coding and
control sequences employs ligation and restriction techniques which
are well understood in the art (Young et al., Nature 316:450-452
(1988)). Double-stranded cDNA encoding delta opioid receptor
protein is synthesized and prepared for insertion into a plasmid
vector CDM8. Alternatively, vectors such as Bluescript.sup.2 or
Lambda ZAP.sup.2 (Stratagene, San Diego, Calif.) or a vector from
Clontech, Palo Alto, Calif., can be used in accordance with
standard procedures (see, e.g., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, Sambrook, et al., eds. second edition
(1989)).
Site specific DNA cleavage is performed by treating with the
suitable restriction enzyme, such as EcoRI (or enzymes), under
conditions which are generally understood in the art, and the
particulars of which are specified by the manufacturer of these
commercially available restriction enzymes. See, e.g., New England
Biolabs, Product Catalog. In general, about 1 .mu.g of DNA is
cleaved by one unit of enzyme in about 20 .mu.l of buffer solution;
in the examples herein, typically, an excess of restriction enzyme
is used to ensure complete digestion of the DNA substrate.
Incubation times of about one hour to two hours at about 37.degree.
C. are workable, although variations can be tolerated. After each
incubation, protein is removed by extraction with
phenol/chloroform, and can be followed by other extraction and the
nucleic acid recovered from aqueous fractions by precipitation with
ethanol.
In vector construction employing "vector fragments", the vector
fragment is commonly treated with bacterial alkaline phosphatase
(BAP) or calf intestinal alkaline phosphatase (CIP) in order to
remove the 5' phosphate and prevent religation of the vector.
Digestions are conducted at pH 8 in approximately 150 mM Tris, in
the presence of Na.sup.+ and Mg.sup.++ using about 1 unit of BAP or
CIP per .mu.g of vector at 60.degree. C. or 37.degree. C.,
respectively, for about one hour. In order to recover the nucleic
acid fragments, the preparation is extracted with phenol/chloroform
and ethanol precipitated. Alternatively, religation can be
prevented in vectors which have been double digested by additional
restriction enzyme digestion of the unwanted fragments.
Ligations are performed in 15-50 .mu.l volumes under the following
standard conditions and temperatures: 20 mM Tris-HCl, pH 7.5, 10 mM
MgCl.sub.2, 10 mM DTT, 33 .mu.g/ml BSA, 10 mM to 50 mM NaCl, and
either 40 .mu.M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at
0.degree. C. (for "sticky end" ligation) or 1 mM ATP, 0.3-0.6
(Weiss) units T4 DNA ligase at 14.degree. C. (for "blunt end"
ligation). Intermolecular "sticky end" ligations are usually
performed at 33-100 .mu.g/ml total DNA concentrations (5-100 nM
total end concentration). Intermolecular blunt end ligations
(usually employing a 10-30 fold molar excess of linkers) are
performed at 1 .mu.M total ends concentration.
Correct ligations for vector construction are confirmed according
to the procedures of Young et al., Nature, 316:450-452 (1988).
cDNA Library Screening
cDNA libraries can be screened using reduced stringency conditions
as described by Ausubel et al., Current Protocols in Molecular
Biology, Greene Publishing and Wiley-Interscience, New York (1990),
or by using methods described in Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, Sambrook et al., eds., second edition
(1989), or by using a colony or plaque hybridization procedure with
a fragment of the DOR-1 cDNA coding for delta opioid receptor
protein.
Plaque hybridization is typically carried out as follows: Host
bacteria such as LE 392 (Stratagene) are grown overnight at
37.degree. in LB Broth (Molecular Cloning: A Laboratory Manual,
supra), gently pelleted and resuspended in one half the original
volume of 10 mM MgSO.sub.4, 10 mM CaCl.sup.2. After titration, an
amount of the phage library containing approximately 50,000 plaque
forming units (pfu) is added to 300 .mu.l of the host bacteria,
incubated at 37.degree. for 15 minutes and plated onto NZYCM agar
with 10 ml NZYCM top agarose. A total of a million plaques
distributed on 20 fifteen cm plates are screened. For colony
screening, transfected bacteria are plated onto LB broth plates
with the appropriate antibiotics. After the plaques or colonies
have grown to 1 mm, the plates are chilled at 4.degree. C. for at
least two hours, and then overlaid with duplicate nitrocellulose
filters, followed by denaturation of the filters in 0.5 M NaOH/1.5
M NaCl for five minutes and neutralization in 0.5 M Tris, pH
7.4/1.5 M NaCl for five minutes. The filters are then dried in air,
baked at 80.degree. C. for two hours, washed in 5X SSC/0.5% SDS at
68.degree. C. for several hours, and prehybridized in 0.5 M
NaPO.sub.4, pH 7.2/1% BSA/1 mM EDTA/7% SDS/100 .mu.g/ml denatured
salmon sperm DNA for more than 4 hours. Using the DOR-1 cDNA
(described herein) labeled by random priming as a probe, high
stringency hybridization is carried out in the same solution at
68.degree. C., and the temperature is reduced to 50-60.degree. C.
for lower stringency hybridization. After hybridization for 16-24
hours, the filters are washed first in 40 mM NaPO.sub.4, pH
7.2/0.5% BSA/5% SDS/1 mM EDTA twice for one hour each, then in 40
mM NaPO.sub.4, pH 7.2/1% BSA/1 mM EDTA for one hour each, both at
the same temperature as the hybridization (Boulton et al., Cell
65:663-675 (1991)). The filters will then be exposed to film with
an enhancing screen at -70.degree. C. for one day to one week.
Positive signals are then aligned to the plates, and the
corresponding positive phage is purified in subsequent rounds of
screening, using the same conditions as in the primary screen.
Purified phage clones are then used to prepare phage DNA for
subcloning into a plasmid vector for sequence analysis. The various
independent clones are also analyzed in terms of tissue
distribution, using Northern blots and in situ hybridization using
standard methods, as well as in terms of function, using expression
in a heterologous eucaryotic expression system such as COS
cells.
Expression of Delta Opioid Receptor Protein
The complete nucleotide sequence, described herein, encoding delta
opioid receptor protein can be expressed in a variety of systems.
The cDNA can be excised by suitable restriction enzymes and ligated
into procaryotic or eucaryotic expression vectors for such
expression.
For example, as set forth below, the cDNA encoding the protein is
expressed in COS cells. To effect functional expression, the
plasmid expression vector CDM8 (Aruffo and Seed, Proc. Natl. Acad.
Sci. USA 84:8573-8577 (1987), provided by Drs. Aruffo and Seed
(Harvard University, Boston, Mass.) was used. Alternatively, other
suitable expression vectors such as retroviral vectors can be
used.
Both procaryotic and eucaryotic systems can be used to express the
delta opioid receptor; however, eucaryotic hosts are preferred.
Eucaryotic microbes, such as yeast, can be used as hosts for mass
production of the delta opioid receptor protein. Laboratory strains
of Saccharomyces cerevisiae, Baker's yeast, are used most, although
a number of other strains are commonly available. Vectors
employing, for example, the 2.mu. origin of replication of Broach,
Meth. Enz. 101:307 (1983), or other yeast compatible origins of
replications (see, e.g., Stinchcomb et al., Nature 282:39 (1979));
Tschempe et al., Gene 10:157 (1980); and Clarke et al., Meth. Enz.
101:300 (1983)) can be used. Control sequences for yeast vectors
include promoters for the synthesis of glycolytic enzymes (Hess et
al., J. Adv. Enzyme Reg. 7:149 (1968); Holland et al., Biochemistry
17:4900 (1978)). Additional promoters known in the art include the
promoter for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.
Chem. 255:2073 (1980)), and those for other glycolytic enzymes.
Other promoters, which have the additional advantage of
transcription controlled by growth conditions are the promoter
regions for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen
metabolism, and enzymes responsible for maltose and galactose
utilization. It is also believed terminator sequences are desirable
at the 3' end of the coding sequences. Such terminators are found
in the 3' untranslated region following the coding sequences in
yeast-derived genes.
Alternatively, genes encoding delta opioid receptor protein are
expressed in eucaryotic host cell cultures derived from
multicellular organisms. (See, e.g., Tissues Cultures, Academic
Press, Cruz and Patterson, eds. (1973)). These systems have the
additional advantage of the ability to splice out introns, and thus
can be used directly to express genomic fragments. Useful host cell
lines include amphibian oocytes such as Xenopus oocytes, COS cells,
VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and insect
cells such as SF9 cells. Expression vectors for such cells
ordinarily include promoters and control sequences compatible with
mammalian cells such as, for example, the commonly used early and
late promoters from baculovirus, vaccinia virus, Simian Virus 40
(SV40) (Fiers et al., Nature 273:113 (1973)), or other viral
promoters such as those derived from polyoma, Adenovirus 2, bovine
papilloma virus, or avian sarcoma viruses. The controllable
promoter, hMTII (Karin et al., Nature 299:797-802 (1982)) may also
be used. General aspects of mammalian cell host system
transformations have been described by Axel (U.S. Pat. No.
4,399,216 issued Aug. 16, 1983). It now appears, that "enhancer"
regions are important in optimizing expression; these are,
generally, sequences found upstream or downstream of the promoter
region in non-coding DNA regions. Origins of replication can be
obtained, if needed, from viral sources. However, integration into
the chromosome is a common mechanism for DNA replication in
eucaryotes.
If procaryotic systems are used, an intronless coding sequence
should be used, along with suitable control sequences. The cDNA of
delta opioid receptor protein can be excised using suitable
restriction enzymes and ligated into procaryotic vectors along with
suitable control sequences for such expression.
Procaryotes most frequently are represented by various strains of
E. coli; however, other microbial strains may also be used.
Commonly used procaryotic control sequences which are defined
herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding site
sequences, including such commonly used promoters as the
beta-lactamase (penicillinase) and lactose (lac) promoter systems
(Chang et al., Nature 198:1056 (1977)) and the tryptophan (trp)
promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980))
and the lambda derived P.sub.L promoter and N-gene ribosome binding
site (Shimatake et al., Nature 292:128 (1981)).
Depending on the host cell used, transformation is carried out
using standard techniques appropriate to such cells. The treatment
employing calcium chloride, as described by Cohen, Proc. Natl.
Acad. Sci. USA (1972) 69:2110 (1972) or the CaCl.sub.2 method
described in Maniatis, et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Press, Sambrook et al., 2nd edition
(1989)) can be used for procaryotes or other cells which contain
substantial cell wall barriers. For mammalian cells without such
cell walls, the calcium phosphate precipitation method of Graham
and van der Eb, Virology 54:546 (1978), optionally as modified by
Wigler et al., Cell 16:777-785 (1979), or by Chen and Okayama,
supra, can be used. Transformations into yeast can be carried out
according to the method of Van Solingen et al., J. Bact. 130:946
(1977), or of Hsiao et al., Proc. Natl. Acad. Sci. USA 76:3829
(1979).
Other representative transfection methods include viral
transfection, DEAE-dextran mediated transfection techniques,
lysozyme fusion or erythrocyte fusion, scraping, direct uptake,
osmotic or sucrose shock, direct microinjection, indirect
microinjection such as via erythrocyte-mediated techniques, and/or
by subjecting host cells to electric currents. The above list of
transfection techniques is not considered to be exhaustive, as
other procedures for introducing genetic information into cells
will no doubt be developed.
Modulation of Expression by Antisense Sequences
Alternatively, antisense sequences may be inserted into cells
expressing delta opioid receptors as a means to modulate functional
expression of the receptors encoded by sense oligonucleotides. The
antisense sequences are prepared from known sense sequences (either
DNA or RNA), by standard methods known in the art. Antisense
sequences specific for the delta opioid receptor gene or RNA
transcript can be used to bind to or inactivate the
oligonucleotides encoding the delta opioid receptor.
Terminology
As used herein, the singular forms "a", "an" and "the" include
plural reference unless the context clearly dictates otherwise.
Thus, for example, reference to "a receptor" includes mixtures of
such receptors, reference to "an opioid" includes a plurality of
and/or mixtures of such opioids and reference to "the host cell"
includes a plurality of such cells of the same or similar type and
so forth.
Unless defined otherwise all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. The
following examples are intended to illustrate but not to limit the
invention. Temperatures are in .degree. C. and pressures at near
atmospheric unless otherwise specified.
Preparation of Mono .sup.125 I-DADLE
DADLE (Peninsula Laboratories Inc.) was iodinated using the iodogen
method. (Maidment, et al., in Microdialysis in the Neurosciences,
T. Robinson and J. Justice, eds., pp. 275-303 (Elsevier, 1991)).
Both mono- and di-iodinated forms are produced. It has been
reported that di-iodo-DADLE does not bind opiate receptors, due to
the di-iodination of the tyrosine residue. (Miller, R. J., et al.,
Life Sci. 22(5):379-88 (February 1978)). Accordingly,
mono-iodinated DADLE is preferred. Mono-.sup.125 I-DADLE is also
preferred because it has extremely high specific activity compared
to DADLe labeled with other isotopes. Thus, exposure times on the
order of days, rather than weeks or months can be used.
By employing a molar ratio of sodium iodide to peptide of
approximately 1:100 when carrying out iodination, the yield of the
preferred mono-iodinated DADLE was increased. Additionally, to
further enhance the yield of the mono-iodinated form, iodinated
DADLE (containing both mono- and di-iodinated forms) was purified
by reverse-phase HPLC. (Maidment et al., supra) Employing this
procedure a single major radiolabeled peak of the mono-iodinated
DADLE separated from di-iodinated and non-iodinated forms.
DADLE monolabeled with 125I is crucial to successful screening.
Radiolabeled .sup.125 I-DADLE differs from DADLE in several
important parameters--size, hydrophobicity, and binding affinity
(slightly lower). The purification of mono-iodinated from
di-iodinated and non-iodinated DADLE by the HPLC step yields a
ligand with very high specific activity (approximately 2000
Ci/mmol). The specific activity of the mono-iodinated form is
approximately 100 times greater than that obtained by using the
unseparated mixture of mono-, di-, and non-iodinated DADLE.
Monolabeled .sup.125 I-DADLE must be used within a few days of its
preparation.
EXAMPLE 1
Preparation of DOR-1
The NG108-15 cell line (available from Dr. Christopher Evans,
U.C.L.A.) comprises a homogeneous and enriched source of delta
opioid receptors. Utilizing mRNA isolated from NG108-15, a
random-primed, size-selected cDNA library was constructed in
plasmid vector CDM8. The cDNA library was amplified in bacteria.
The cDNA library was transfected into COS-7 cells by
electroporation. Transiently transfected COS lawns were screened
and selected with highly purified mono-.sup.125 I2dAla, 5dLeu
enkephalin (.sup.125 I-DADLE). Positive clones were identified by
film autoradiography, and plasmids from these cells were recovered
and amplified in bacteria. Thereafter, the plasmids were
re-transfected into COS cells. Following three cycles of such
plasmid enrichment, individual clones were transfected and a pure
clone was identified that bound .sup.125 I-DADLE.
A. Construction of the cDNA Library
RNA was prepared from NG108-15 cells by homogenization in 6 M
guanidinium isothiocyanate, followed by centrifugation through
cesium chloride (J. M. Chirgwin, et al., Biochemistry 18:5294
(1979)). Poly-A.sup.+ RNA was isolated by chromatography over
oligo-dT-cellulose (H. Aviv and P. Leder, Proc. Natl. Acad. Sci.
USA 69: 1408 (1972)). Using this RNA as a template, random hexamers
were used to prime cDNA synthesis by avian myeloblastosis virus
reverse transcriptase (Life Sciences Inc.). Second strand synthesis
was accomplished with RNase-H and E. coli DNA polymerase (U. Gubler
and B. J. Hoffman, Gene 24: 263 (1983)). The ends of the cDNAs were
rendered blunt with T4 DNA polymerase and BstXI linkers were added.
cDNA longer than 1.5 kB was selected by electrophoresis through 5%
acrylamide followed by electro-elution. The 1.5 kB cDNA was ligated
to the CDM8 vector (A. Aruffo and B. Seed, Proc. Natl. Acad. Sci.
USA 84: 8573 (1987)), and then transformed into MC-1061 bacteria by
electroporation (W. J. Dower, J. F. Miller and C. W. Ragsdale,
Nucl. Acids Res. 16: 6127 (1988)). Accordingly, six pools of
plasmid DNA were prepared from the original cDNA library of
approximately 2.times.10.sup.6 recombinants.
B. Plasmid Transfection by Electroporation and Expression in COS
cells
COS cells were grown at high density and were harvested in trypsin,
then resuspended at 2.times.10.sup.7 /ml in 1.2X RPMI containing
20% fetal calf serum. These cells were then incubated for ten
minutes at 4.degree. C. with 20 ug recombinant plasmid DNA from the
cDNA library of paragraph A, and then electroporated at 960 uF and
230 V in a 0.4 cm gap cuvette (BioRad). The cells were then
incubated an additional ten minutes at 4.degree. C., and then
plated into Dulbecco's Modified Eagle's Medium (DMEM) plus 10%
fetal calf serum (FCS).
C. Screening of Transfected COS Cells
The transfected COS cells as obtained in paragraph B were grown for
three days, then screened using radiolabeled mono .sup.125 I-DADLE.
Transfected COS lawns were washed with PBS, then incubated at room
temperature with 10-20 nM .sup.125 I-DADLE in KHRB containing 1%
BSA. After 1 hour, the plates were washed rapidly several times
with ice cold PBS then dried on ice with strong flow of forced cold
air. Plates were exposed on Dupont Cronex film in cassettes at room
temperature. Positive clones were identified by careful alignment
of the film with the petri dish via low power microscopy.
DNA was removed from positive cells by solubilization in 0.1% SDS
in TE containing 1 .mu.g/.mu.l tRNA delivered from a syringe
attached to a capillary tube on a micromanipulator. Plasmids were
purified from the extracted cells using the Hirt lysis procedure
(Hirt, B., J. Mol. Biol. 26: 365-369 (1967)), and electroporated
into MC-1061 bacteria. The plasmids were purified then
retransfected into COS cells. Following three such enriching
cycles, individual plasmid clones were transfected into COS cells
yielding a single clone, named the DOR-1 clone.
EXAMPLE 2
Characterization of DOR-1
The DOR-1 clone initially was characterized by screening cell
membrane fractions, from cells expressing DOR-1, with the labelled
DADLE it was found that binding of .sup.125 I DADLE was displaced
by nanomolar concentrations of opiate alkaloids diprenorphine,
morphine, etorphine, and by DADLE, DSLET and DPDPE. Dextrophan (10
.mu.M) did not displace the .sup.125 I DADLE, whereas its
opioid-active enantiomer levorphanol did displace the radiolabeled
DADLE (data not illustrated). Additionally, the .mu.-selective
ligand DAGO (5 .mu.M) did not displace the counts (data not
illustrated).
The DOR-1 clone was further characterized pharmacologically by
assessing binding of .sup.3 H-diprenorphine to intact cells
expressing the DOR-1 clone (FIG. 1), and by assessing displacement
of .sup.3 H-diprenorphine from membrane fractions of such cells
(FIGS. 2 and 3).
Binding assays were conducted on intact cells in KRHB, 1% BSA; or
on membranes in 25 mM HEPES, 5 mM MgCl.sub.2 pH 7.7. Cells were
harvested with PBS containing 1 mM EDTA, washed 2.times. with PBS
then resuspended in KHRB. Membranes prepared from the cells (Law P.
Y. E, et al., Mol. Pharm. 23: 26-35 (1983)) were used directly in
the binding assay. Binding assays were conducted in 96 well
polypropylene cluster plates (Costar), at 4.degree. C. in a total
volume of 100 .mu.l with an appropriate amount of radiolabeled
ligand. Following 1 hour of incubation, plates were harvested on a
Tomtec harvester and "B" type filtermats were counted in a
Betaplate (Pharmacia) scintillation counter using Meltilex B/HS
(Pharmacia) melt-on scintillator sheets.
Intact cells expressing DOR-1 were analyzed with the high affinity
opiate antagonist .sup.3 H-diprenorphine. Specific binding was
defined by the counts displaced by 400 nM Diprenorphine. FIG. 1
shows a saturation curve for .sup.3 H-diprenorphine for NG108-15
cells, and COS-7 cells transfected with the delta opioid receptor
clone. Untransfected COS cells, or COS cells transfected with
plasmid having no insert showed no specific binding. Thus, it was
seen that the opioid binding of COS-DOR-1 cells is similar to that
of NG108-15 cells.
Membranes prepared by standard methods from transfected COS-7 cells
were employed for a more extensive pharmacological characterization
of the receptor encoded by the DOR-1 clone. The affinities for the
alkaloid opiates in competition for .sup.3 H-diprenorphine are
illustrated in FIG. 2, and the affinities for the opioid peptides
in competition for .sup.3 H-diprenorphine are set forth in FIG.
3.
The alkaloid opiates tested in FIG. 2 were unlabeled diprenorphine,
a high affinity antagonist for delta receptors; etorphine, a high
affinity agonist for delta, mu and kappa receptors; levorphanol, a
low affinity agonist for delta receptors; morphine, a low affinity
agonist for delta receptors and a high affinity agonist for mu
receptors; and dextrorphan, a non-opiate active enantiomer of
levorphanol which should not bind delta receptors. As shown in FIG.
2, the displacement of .sup.3 H-diprenorphine, in decreasing order
of affinity, was observed with diprenorphine, etorphine,
levorphanol and morphine. As expected, .sup.3 H-diprenorphine was
not displaced by dextrorphan.
The opioid peptides tested in FIG. 3 were DADLE, a high affinity
agonist for mu and delta receptors; DSLET and DPDPE, both high
affinity agonists of delta (but not mu) receptors; DAGO, a
selective agonist for mu receptors; and Dynorphin 1-17, a high
affinity agonist for kappa receptors and moderate to low affinity
agonist for delta receptors. As shown in FIG. 3, the displacement
of .sup.3 H-diprenorphine, in decreasing order of affinity, was
observed for DSLET, DPDPE and DADLE, and Dynorphin 1-17. Only weak
displacement by DAGO was observed.
Thus, the rank order of the alkaloid opiate and opioid peptide
affinities and selectivities for the cloned delta opioid receptor
support the hypothesis that the DOR-1 clone encodes a delta opioid
receptor.
EXAMPLE 3
Northern Blot Analysis of RNA
For Northern analysis, the mRNA from NG108-15 cells, and from cells
dissected from regions of rat brain was separated by
electrophoresis through 2.2 M formaldehyde/1.5% agarose, blotted to
nylon and hybridized in aqueous solution at high stringency. The
filters were prehybridized in 0.5 M NaPO.sub.4, pH 7.2; 1% BSA; 1
mM EDTA; 7% SDS; and 100 ug/ml denatured salmon sperm DNA for at
least four hours at 68.degree. C. (T. G. Boulton et al., Cell 65:
663 (1991)). The filters were then hybridized overnight under these
same conditions with .gtoreq.5.times.10.sup.6 cpm/ml purified cDNA
insert labelled by random priming (A. P. Feinberg and B.
Vogelstein, Anal. Biochem. 132: 6 (1983)). The filters were twice
washed in 40 mM NaPO.sub.4, pH 7.2; 0.5% BSA; 5% SDS; and 1 mM EDTA
for one hour, and then washed twice in 40 mM NaPO.sub.4, pH 7.2; 1%
SDS; and 1 mM EDTA for one hour each, all at 68.degree. C.
Thereafter autoradiography was performed with DuPont Cromex
Lightening Plus at -70.degree. C.
The results of the Northern analysis of the mRNA showed the
presence of multiple bands hybridizing to the probe at
approximately 8.7, 6.8, 4.4, 2.75 and 2.2 kilobases (Kb) (FIG. 4).
Also, the Northern analysis indicates that the pattern of mRNA may
vary between brain regions. At present, it is unclear whether these
mRNAs encode different protein sequences, and if so, whether these
messages represent different types or sub-types of opioid
receptors.
EXAMPLE 4
Southern Blot Analysis of DNA
The radiolabeled DOR-1 cDNA probe was hybridized to genomic
Southern blots by standard methods. (Sambrook, supra) Accordingly,
the radiolabeled DOR-1 cDNA probe was hybridized under high
stringency conditions to a blot of NG108-15, mouse, rat and human
DNA cut with restriction endonuclease Bam HI. (FIG. 7) Single bards
were observed in the clones containing the NG108-15, mouse, and rat
DNA. The sizes of the bands hybridizing to the cDNA probe estimated
to be 5.2 kB (NG108-15), 5.2 kB (mouse), and 5.7 kB (rat). These
data manifest the close homology of the mouse and rat genes, and
also demonstrate that the DOR-1 clone is from the murine parent of
the NG108-15 cell line.
In a blot containing EcoRI-cut genomic DNA from many different
species (data not illustrated), hybridization of the DOR-1 cDNA
under conditions of moderate stringency showed two bands in each
lane of mouse, rat, human, rabbit, and several other mammalian
species. This demonstrates that the opioid receptor gene in all of
these species is closely related. Further, these data show that the
genes or cDNAs from each of these species may readily be cloned
using hybridization under moderate stringency.
EXAMPLE 5
Determination of the cDNA Sequence
Isolated cDNA encoding the delta opioid receptor was analyzed by
subcloning the insert from the cDNA clone into a plasmid such as
pBluescript.TM. (Stratagene, San Diego, Calif.) and using the
dideoxy method (Sanger et al., Proc. Natl. Acad. Sci. USA
74:5463-5467 (1977)). The sequence of the cDNA was determined from
single-stranded DNA and specifically designed internal primers,
using both Sequenase and .DELTA.Taq cycle sequencing kits (USB).
These kits utilize the dideoxy chain termination method and are
widely used in the art. The DNA sequence and predicted protein
sequence was then compared to established databanks such as
GenBank.
By sequencing the cDNA insert in the DOR-1 clone, an open reading
frame of 370 amino acids was revealed. (FIG. 5) Comparisons with
known sequences in GenBank showed highest homology between DOR-1
and the G-protein-coupled receptor for somatostatin (57% amino acid
identity), and slightly lower homology with the receptor binding
angiotensin, the two chemotactic factors IL-8 and N-formyl peptide.
FIG. 6 shows the homology to the human somatostatin 1 receptor. The
close homology of the present delta-receptor clone with the
somatostatin receptor is especially noteworthy since somatostatin
ligands are reported to bind to opioid receptors, and to have
molecular mechanisms similar to those in delta receptors.
Other features of the DOR-1 clone deduced from the cDNA sequence
include three consensus glycosylation sites at residues 18 and 33
(predicted to be in the extracellular N-terminal domain), and at
residue 310 (close to the C-terminus and predicted to be
intracellular). Phosphokinase C consensus sites are present within
predicted intracellular domains, at residues 242, 255, 344, and
352. Seven putative membrane-spanning regions were identified based
on hydrophobicity profiles, as well as homology with Rhodopsin and
other G-protein coupled receptors which have been analyzed with
respect to membrane-spanning regions using MacVector (I.B.I.)
analysis. The DOR-1 clone isolated in accordance with the
principles of the present invention produces a delta receptor with
a predicted molecular weight of 40,558 daltons prior to
post-translational modifications such as N-glycosylation.
EXAMPLE 6
Isolation of Opioid Receptor Genomic Clones
Genomic clone isolation was carried out according to techniques
known in the art. To isolate opiate receptor genomic clones,
300,000 human genomic clones in .gamma. gem 11 (Promega) and a
similar number of mouse genomic clones in lambda Fix (Stratagene)
were plated on host strain Le392 and probed with the 1.1 kB delta
opiate receptor pst/xba 1 fragment, obtained by standard methods.
It has been determined by standard methodology that the 1.1 Kb
probe primarily contains coding region. The conditions for
hybridization were of fairly low stringency-50% formamide/6XSSC,
overnight at 37.degree. C. The washes were performed also at low
stringency-2X SSC, 0.1% SDS at room temperature.
One mouse clone and 4 human genomic clones were isolated and
purified by sequential rounds of hybridization and plaque
purification. DNA preparation and restriction analysis showed that
the four human clones had very different Eco R1 digestion patterns.
The 1.1 kB opiate receptor probe hybridized to a different single
Eco R1 band in Southern blot analysis. (data not shown).
The genomic clones were digested into smaller fragments by Eco R1
and Taq 1, then shotgun cloned into the appropriate site of
Bluescript. The sequence of these human genomic clones appears very
similar to the original mouse cDNA, but some divergences are
apparent. Such divergences are common within species, and often
occur in regions that are not essential for function.
Accordingly, a human genomic clone has been isolated (DOR-h1) that
encodes a delta opioid receptor. A partial sequence of Human
Genomic Clone DOR-h1 reads:
-CAC TCT TGC ATT GCT CTA GGT TAC ACA (SEQ ID NO:7) - AAC AGC TGC
CTC AAC CCA GTC CTT TAT - GCA TTT CTG GAT GAA AAC TTC AAA CGA - TGC
TTC AGA-)
Seventy out of ninety nucleotides of the human DNA (DOR-h1) match
exactly with DOR-1 DNA near the 3' end of the mouse cDNA.
When the amino acid sequence encoded by these DNA sequences are
compared, it is found that there is 27 out of 30 amino acid
identity:
DOR-1: H L C I A L G Y A N S S L N P V L Y A F L D E N F K R C F R
(SEQ ID NO:8) .vertline. .vertline. .vertline. .vertline.
.vertline. .vertline. .vertline. .vertline. .vertline. .vertline.
.vertline. .vertline. .vertline. .vertline. .vertline. .vertline.
.vertline. .vertline. .vertline. .vertline. .vertline. .vertline.
.vertline. .vertline. .vertline. .vertline. .vertline. DOR-h1: H F
C I A L G Y T N S C L N P V L Y A F L D E N F K R C F R (SEQ ID
NO:9)
EXAMPLE 7
Isolation of Delta Opioid Receptor Clones From Additional
Organisms
In order to isolate the delta opioid receptor from mammalian brain
cells, for example human brain cells, a random-primed human
brainstem cDNA library in lambda Zap (Stratagene) was screened
using the murine cDNA encoding the DOR-1 described herein. Positive
plaques were purified and rescreened. Individual positive clones
are sequenced and characterized as above.
EXAMPLE 8
Determination of Probable Antigenic Sequences
By evaluating the amino acid sequence of the DOR-1 delta opioid
receptor with the MacVector (I.B.I.) antigenic index, and the
antigenic index in accordance to Jameson, B. and H. Wolf, Comput.
Applic. in the Biosciences, 4, 181-186 (1988), the following
underlined sequences of the delta opioid receptor were determined
to have a high antigenic potential:
(SEQ ID NO:10) NH.sub.2
MELVPSARAELOSSPLVNLSDAFPSAFPSAGANASGSPGARSAS -
SLALAIAITALYSAVCAVGLLGNVLVMFGIVRYTKLKTATNIYIFNL -
ALADALATSTLPFQSAKYLMETWPFGELLCKAVLSIDYYNMFTSIFT -
LTMMSVDRYIAVCHPVKALDFRTPAKAKLINICIWVLASGVGVPIMV -
MAVTQPRDGAVVCMLQFPSPSWYWDTVTKICVFLFAFVVPILIITVC -
YGLMLLRLRSVRLLSGSKEKDRSLRRITRMVLVVVGAFVVCWAPIHI -
FVIVWTLVDINRRDPLVVAALHLCIALGYANSSLNPVLYAFLDENFK -
RCFRQLCRTPCGRQEPGSLRRPRQATTRERVTACTPSDGPGGGAAA- - COOH.
The N-terminal sequence is extracellular, the other four sequences
are predicted to be intracellular.
__________________________________________________________________________
# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii)
NUMBER OF SEQUENCES: 15 - - - - (2) INFORMATION FOR SEQ ID NO:1: -
- (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1829 base - #pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -
- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 29..1144 - - (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:1: - - GCACGGTGGA GACGGACACG
GCGGCGCC ATG GAG CTG GTG CCC - #TCT GCC CGT 52 - # Met Glu Leu -
#Val Pro Ser Ala Arg - # 1 - # 5 - - GCG GAG CTG CAG TCC TCG CCC
CTC GTC AAC CT - #C TCG GAC GCC TTT CCC 100 Ala Glu Leu Gln Ser Ser
Pro Leu Val Asn Le - #u Ser Asp Ala Phe Pro 10 - # 15 - # 20 - -
AGC GCC TTC CCC AGC GCG GGC GCC AAT GCG TC - #G GGG TCG CCG GGA GCC
148 Ser Ala Phe Pro Ser Ala Gly Ala Asn Ala Se - #r Gly Ser Pro Gly
Ala 25 - # 30 - # 35 - # 40 - - CGT AGT GCC TCG TCC CTC GCC CTA GCC
ATC GC - #C ATC ACC GCG CTC TAC 196 Arg Ser Ala Ser Ser Leu Ala Leu
Ala Ile Al - #a Ile Thr Ala Leu Tyr 45 - # 50 - # 55 - - TCG GCT
GTG TGC GCA GTG GGG CTT CTG GGC AA - #C TGT CTC GTC ATG TTT 244 Ser
Ala Val Cys Ala Val Gly Leu Leu Gly As - #n Cys Leu Val Met Phe 60
- # 65 - # 70 - - GGC ATC GTC CGG TAC ACC AAA TTG AAG ACC GC - #C
ACC AAC ATC TAC ATC 292 Gly Ile Val Arg Tyr Thr Lys Leu Lys Thr Al
- #a Thr Asn Ile Tyr Ile 75 - # 80 - # 85 - - TTC AAT CTG GCT TTG
GCT GAT GCG CTG GCC AC - #C AGC ACG CTG CCC TTC 340 Phe Asn Leu Ala
Leu Ala Asp Ala Leu Ala Th - #r Ser Thr Leu Pro Phe 90 - # 95 - #
100 - - CAG AGC GCC AAG TAC TTG ATG GAA ACG TGG CC - #G TTT GGC GAG
CTG CTG 388 Gln Ser Ala Lys Tyr Leu Met Glu Thr Trp Pr - #o Phe Gly
Glu Leu Leu 105 1 - #10 1 - #15 1 - #20 - - TGC AAG GCT GTG CTC TCC
ATT GAC TAC TAC AA - #C ATG TTC ACT AGC ATC 436 Cys Lys Ala Val Leu
Ser Ile Asp Tyr Tyr As - #n Met Phe Thr Ser Ile 125 - # 130 - # 135
- - TTC ACC CTC ACC ATG ATG AGC GTG GAC CGC TA - #C ATT GCT GTC TGC
CAT 484 Phe Thr Leu Thr Met Met Ser Val Asp Arg Ty - #r Ile Ala Val
Cys His 140 - # 145 - # 150 - - CCT GTC AAA GCC CTG GAC TTC CGG ACA
CCA GC - #C AAG GCC AAG CTG ATC 532 Pro Val Lys Ala Leu Asp Phe Arg
Thr Pro Al - #a Lys Ala Lys Leu Ile 155 - # 160 - # 165 - - AAT ATA
TGC ATC TGG GTC TTG GCT TCA GGT GT - #C GGG GTC CCC ATC ATG 580 Asn
Ile Cys Ile Trp Val Leu Ala Ser Gly Va - #l Gly Val Pro Ile Met 170
- # 175 - # 180 - - GTC ATG GCA GTG ACC CAA CCC CGG GAT GGT GC - #A
GTG GTA TGC ATG CTC 628 Val Met Ala Val Thr Gln Pro Arg Asp Gly Al
- #a Val Val Cys Met Leu 185 1 - #90 1 - #95 2 - #00 - - CAG TTC
CCC AGT CCC AGC TGG TAC TGG GAC AC - #T GTG ACC AAG ATC TGC 676 Gln
Phe Pro Ser Pro Ser Trp Tyr Trp Asp Th - #r Val Thr Lys Ile Cys 205
- # 210 - # 215 - - GTG TTC CTC TTT GCC TTC GTG GTG CCG ATC CT - #C
ATC ATC ACG GTG TGC 724 Val Phe Leu Phe Ala Phe Val Val Pro Ile Le
- #u Ile Ile Thr Val Cys 220 - # 225 - # 230 - - TAT GGC CTC ATG
CTA CTG CGC CTG CGC AGC GT - #G CGT CTG CTG TCC GGT 772 Tyr Gly Leu
Met Leu Leu Arg Leu Arg Ser Va - #l Arg Leu Leu Ser Gly 235 - # 240
- # 245 - - TCC AAG GAG AAG GAC CGC AGC CTG CGG CGC AT - #C ACG CGC
ATG GTG CTG 820 Ser Lys Glu Lys Asp Arg Ser Leu Arg Arg Il - #e Thr
Arg Met Val Leu 250 - # 255 - # 260 - - GTG GTG GTG GGC GCC TTC GTG
GTG TGC TGG GC - #G CCC ATC CAC ATC TTC 868 Val Val Val Gly Ala Phe
Val Val Cys Trp Al - #a Pro Ile His Ile Phe 265 2 - #70 2 - #75 2 -
#80 - - GTC ATC GTC TGG ACG CTG GTG GAC ATC AAT CG - #G CGC GAC CCA
CTT GTG 916 Val Ile Val Trp Thr Leu Val Asp Ile Asn Ar - #g Arg Asp
Pro Leu Val 285 - # 290 - # 295 - - GTG GCC GCA CTG CAC CTG TGC ATT
GCG CTG GG - #C TAC GCC AAC AGC AGC 964 Val Ala Ala Leu His Leu Cys
Ile Ala Leu Gl - #y Tyr Ala Asn Ser Ser 300 - # 305 - # 310 - - CTC
AAC CCG GTT CTC TAC GCC TTC CTG GAC GA - #G AAC TTC AAG CGC TGC
1012 Leu Asn Pro Val Leu Tyr Ala Phe Leu Asp Gl - #u Asn Phe Lys
Arg Cys 315 - # 320 - # 325 - - TTC CGC CAG CTC TGT CGC ACG CCC TGC
GGC CG - #C CAA GAA CCC GGC AGT 1060 Phe Arg Gln Leu Cys Arg Thr
Pro Cys Gly Ar - #g Gln Glu Pro Gly Ser 330 - # 335 - # 340 - - CTC
CGT CGT CCC CGC CAG GCC ACC ACG CGT GA - #G CGT GTC ACT GCC TGC
1108 Leu Arg Arg Pro Arg Gln Ala Thr Thr Arg Gl - #u Arg Val Thr
Ala Cys 345 3 - #50 3 - #55 3 - #60 - - ACC CCC TCC GAC GGC CCG GGC
GGT GGC GCT GC - #C GCC TGACCTACCC 1154 Thr Pro Ser Asp Gly Pro Gly
Gly Gly Ala Al - #a Ala 365 - # 370 - - GACCTTCCCC TTAAACGCCC
CTCCCAAGTG AAGTGATCAG AGGCCACACC GA - #GCTCCCTG 1214 - - GGAGGCTGTG
GCCACCACCA GGACAGCTAG AATTGGGCCT GCACAGAGGG GA - #GGCCTCCT 1274 - -
GTGGGGACGG GCCTGAGGGA TCAAAGGCTC CAGGTTGGAA CGGTGGGGGT GA -
#GGAAGCAG 1334 - - AGCTGGTGAT TCCTAAACTG TATCCATTAG TAAGGCCTCT
CAATGGGACA GA - #GCCTCCGC 1394 - - CTTGAGATAA CATCGGGTTC TGGCCTTTTT
GAACACCCAG CTCCAGTCCA AG - #ACCCAAGG 1454 - - ATTCCAGCTC CAGAACCAGG
AGGGGCAGTG ATGGGGTCGA TGATTTGGTT TG - #GCTGAGAG 1514 - - TCCCAGCATT
TGTGTTATGG GGAGGATCTC TCATCTTAGA GAAGAAAGGG GA - #CAGGGCAT 1574 - -
TCAGGCAAGG CAGCTTGGGG TTTGGTCAGG AGATAAGCGC CCCCCTTCCC TT -
#GGGGGGAG 1634 - - GATAAGTGGG GGATGGTCAC GTTGGAGAAG AGTCAAAGTT
CTCACCACCT TT - #CTAACTAC 1694 - - TCAGCTAAAC TCGTTGAGGC TAGGGCCAAC
GTGACTTCTC TGTAGAGAGG TA - #CAAGCCGG 1754 - - GCCTGATGGG GCAGGCCTGT
GTAATCCCAG TCATAGTGGA GGCTGAGGCT GG - #AAAATTAA 1814 - - GGACCAACAG
CCCGG - # - # - # 1829 - - - - (2) INFORMATION FOR SEQ ID NO:2: - -
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 372 amino - #acids (B)
TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE:
protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - Met Glu Leu
Val Pro Ser Ala Arg Ala Glu Le - #u Gln Ser Ser Pro Leu 1 5 - # 10
- # 15 - - Val Asn Leu Ser Asp Ala Phe Pro Ser Ala Ph - #e Pro Ser
Ala Gly Ala 20 - # 25 - # 30 - - Asn Ala Ser Gly Ser Pro Gly Ala
Arg Ser Al - #a Ser Ser Leu Ala Leu 35 - # 40 - # 45 - - Ala Ile
Ala Ile Thr Ala Leu Tyr Ser Ala Va - #l Cys Ala Val Gly Leu 50 - #
55 - # 60 - - Leu Gly Asn Cys Leu Val Met Phe Gly Ile Va - #l Arg
Tyr Thr Lys Leu 65 - # 70 - # 75 - # 80 - - Lys Thr Ala Thr Asn Ile
Tyr Ile Phe Asn Le - #u Ala Leu Ala Asp Ala 85 - # 90 - # 95 - -
Leu Ala Thr Ser Thr Leu Pro Phe Gln Ser Al - #a Lys Tyr Leu Met Glu
100 - # 105 - # 110 - - Thr Trp Pro Phe Gly Glu Leu Leu Cys Lys Al
- #a Val Leu Ser Ile Asp 115 - # 120 - # 125 - - Tyr Tyr Asn Met
Phe Thr Ser Ile Phe Thr Le - #u Thr Met Met Ser Val 130 - # 135 - #
140 - - Asp Arg Tyr Ile Ala Val Cys His Pro Val Ly - #s Ala Leu Asp
Phe Arg 145 1 - #50 1 - #55 1 - #60 - - Thr Pro Ala Lys Ala Lys Leu
Ile Asn Ile Cy - #s Ile Trp Val Leu Ala 165 - # 170 - # 175 - - Ser
Gly Val Gly Val Pro Ile Met Val Met Al - #a Val Thr Gln Pro Arg 180
- # 185 - # 190 - - Asp Gly Ala Val Val Cys Met Leu Gln Phe Pr - #o
Ser Pro Ser Trp Tyr 195 - # 200 - # 205 - - Trp Asp Thr Val Thr Lys
Ile Cys Val Phe Le - #u Phe Ala Phe Val Val 210 - # 215 - # 220 - -
Pro Ile Leu Ile Ile Thr Val Cys Tyr Gly Le - #u Met Leu Leu Arg Leu
225 2 - #30 2 - #35 2 - #40 - - Arg Ser Val Arg Leu Leu Ser Gly Ser
Lys Gl - #u Lys Asp Arg Ser Leu 245 - # 250 - # 255 - - Arg Arg Ile
Thr Arg Met Val Leu Val Val Va - #l Gly Ala Phe Val Val 260 - # 265
- # 270 - - Cys Trp Ala Pro Ile His Ile Phe Val Ile Va - #l Trp Thr
Leu Val Asp 275 - # 280 - # 285 - - Ile Asn Arg Arg Asp Pro Leu Val
Val Ala Al - #a Leu His Leu Cys Ile 290 - # 295 - # 300 - - Ala Leu
Gly Tyr Ala Asn Ser Ser Leu Asn Pr - #o Val Leu Tyr Ala Phe 305 3 -
#10 3 - #15 3 - #20 - - Leu Asp Glu Asn Phe Lys Arg Cys Phe Arg Gl
- #n Leu Cys Arg Thr Pro 325 - # 330 - # 335 - - Cys Gly Arg Gln
Glu Pro Gly Ser Leu Arg Ar - #g Pro Arg Gln Ala Thr 340 - # 345 - #
350 - - Thr Arg Glu Arg Val Thr Ala Cys Thr Pro Se - #r Asp Gly Pro
Gly Gly 355 - # 360 - # 365 - - Gly Ala Ala Ala 370 - - - - (2)
INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 369 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:3: - - Met Glu Leu Thr Ser Glu Gln Phe Asn Gly Se - #r Gln Val
Trp Ile Pro 1 5 - # 10 - # 15 - - Ser Pro Phe Asp Leu Asn Gly Ser
Leu Gly Pr - #o Ser Asn Gly Ser Asn 20 - # 25 - # 30 - - Gln Thr
Glu Pro Tyr Tyr Asp Met Thr Ser As - #n Ala Val Leu Thr Phe 35 - #
40 - # 45 - - Ile Tyr Phe Val Val Cys Val Val Gly Leu Cy - #s Gly
Asn Thr Leu Val 50 - # 55 - # 60 - - Ile Tyr Val Ile Leu Arg Tyr
Ala Lys Met Ly - #s Thr Ile Thr Asn Ile 65 - #70 - #75 - #80 - -
Tyr Ile Leu Asn Leu Ala Ile Ala Asp Glu Le - #u Phe Met Leu Gly Leu
85 - # 90 - # 95 - - Pro Phe Leu Ala Met Gln Val Ala Leu Val Hi -
#s Trp Pro Phe Gly Lys 100 - # 105 - # 110 - - Ala Ile Cys Arg Val
Val Met Thr Val Asp Gl - #y Ile Asn Gln Phe Thr 115 - # 120 - # 125
- - Ser Ile Phe Cys Leu Thr Val Met Ser Ile As - #p Arg Tyr Leu Ala
Val 130 - # 135 - # 140 - - Val His Pro Ile Lys Ser Ala Lys Trp Arg
Ar - #g Pro Arg Thr Ala Lys 145 1 - #50 1 - #55 1 - #60 - - Met Ile
Asn Val Ala Val Trp Gly Val Ser Le - #u Leu Val Ile Leu Pro 165 - #
170 - # 175 - - Ile Met Ile Tyr Ala Gly Leu Arg Ser Asn Gl - #n Trp
Gly Arg Ser Ser 180 - # 185 - # 190 - - Cys Thr Ile Asn Trp Pro Gly
Glu Ser Gly Al - #a Trp Tyr Thr Gly Phe 195 - # 200 - # 205 - - Ile
Ile Tyr Ala Phe Ile Leu Gly Phe Leu Va - #l Pro Leu Thr Ile Ile 210
- # 215 - # 220 - - Cys Leu Cys Tyr Leu Phe Ile Ile Ile Lys Va - #l
Lys Ser Ser Gly Ile 225 2 - #30 2 - #35 2 - #40 - - Arg Val Gly Ser
Ser Lys Arg Lys Lys Ser Gl - #u Lys Lys Val Thr Arg 245 - # 250 - #
255 - - Met Val Ser Ile Val Val Ala Val Phe Ile Ph - #e Cys Trp Leu
Pro Phe 260 - # 265 - # 270 - - Tyr Ile Phe Asn Val Ser Ser Val Ser
Val Al - #a Ile Ser Pro Thr Pro 275 - # 280 - # 285 - - Ala Leu Lys
Gly Met Phe Asp Phe Val Val Il - #e Leu Thr Tyr Ala Asn 290 - # 295
- # 300 - - Ser Cys Ala Asn Pro Ile Leu Tyr Ala Phe Le - #u Ser Asp
Asn Phe Lys 305 3 - #10 3 - #15 3 - #20
- - Lys Ser Phe Gln Asn Val Leu Cys Leu Val Ly - #s Val Ser Gly Ala
Glu 325 - # 330 - # 335 - - Asp Gly Glu Arg Ser Asp Ser Lys Gln Asp
Ly - #s Ser Arg Leu Asn Glu 340 - # 345 - # 350 - - Thr Thr Glu Thr
Gln Arg Thr Leu Leu Asn Gl - #y Asp Leu Gln Thr Ser 355 - # 360 - #
365 - - Ile - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino - #acids (B) TYPE:
amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:4: - - Tyr Gly Gly Phe Xaa 1 5 - -
- - (2) INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 5 amino - #acids (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:5: - - Tyr Gly Gly Phe Met 1 5 - - - - (2)
INFORMATION FOR SEQ ID NO:6: - - (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 5 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:6: - - Tyr Gly Gly Phe Leu 1 5 - - - - (2) INFORMATION FOR SEQ
ID NO:7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 90 base -
#pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)
TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: - -
CACTCTTGCA TTGCTCTAGG TTACACAAAC AGCTGCCTCA ACCCAGTCCT TT -
#ATGCATTT 60 - - CTGGATGAAA ACTTCAAACG ATGCTTCAGA - # - # 90 - - -
- (2) INFORMATION FOR SEQ ID NO:8: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 30 amino - #acids (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:8: - - His Leu Cys Ile Ala Leu Gly Tyr Ala
Asn Se - #r Ser Leu Asn Pro Val 1 5 - # 10 - # 15 - - Leu Tyr Ala
Phe Leu Asp Glu Asn Phe Lys Ar - #g Cys Phe Arg 20 - # 25 - # 30 -
- - - (2) INFORMATION FOR SEQ ID NO:9: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 30 amino - #acids (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:9: - - His Phe Cys Ile Ala Leu Gly Tyr Thr
Asn Se - #r Cys Leu Asn Pro Val 1 5 - # 10 - # 15 - - Leu Tyr Ala
Phe Leu Asp Glu Asn Phe Lys Ar - #g Cys Phe Arg 20 - # 25 - # 30 -
- - - (2) INFORMATION FOR SEQ ID NO:10: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 372 amino - #acids (B) TYPE: amino
acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:10: - - Met Glu Leu Val Pro Ser Ala
Arg Ala Glu Le - #u Gln Ser Ser Pro Leu 1 5 - # 10 - # 15 - - Val
Asn Leu Ser Asp Ala Phe Pro Ser Ala Ph - #e Pro Ser Ala Gly Ala 20
- # 25 - # 30 - - Asn Ala Ser Gly Ser Pro Gly Ala Arg Ser Al - #a
Ser Ser Leu Ala Leu 35 - # 40 - # 45 - - Ala Ile Ala Ile Thr Ala
Leu Tyr Ser Ala Va - #l Cys Ala Val Gly Leu 50 - # 55 - # 60 - -
Leu Gly Asn Val Leu Val Met Phe Gly Ile Va - #l Arg Tyr Thr Lys Leu
65 - #70 - #75 - #80 - - Lys Thr Ala Thr Asn Ile Tyr Ile Phe Asn Le
- #u Ala Leu Ala Asp Ala 85 - # 90 - # 95 - - Leu Ala Thr Ser Thr
Leu Pro Phe Gln Ser Al - #a Lys Tyr Leu Met Glu 100 - # 105 - # 110
- - Thr Trp Pro Phe Gly Glu Leu Leu Cys Lys Al - #a Val Leu Ser Ile
Asp 115 - # 120 - # 125 - - Tyr Tyr Asn Met Phe Thr Ser Ile Phe Thr
Le - #u Thr Met Met Ser Val 130 - # 135 - # 140 - - Asp Arg Tyr Ile
Ala Val Cys His Pro Val Ly - #s Ala Leu Asp Phe Arg 145 1 - #50 1 -
#55 1 - #60 - - Thr Pro Ala Lys Ala Lys Leu Ile Asn Ile Cy - #s Ile
Trp Val Leu Ala 165 - # 170 - # 175 - - Ser Gly Val Gly Val Pro Ile
Met Val Met Al - #a Val Thr Gln Pro Arg 180 - # 185 - # 190 - - Asp
Gly Ala Val Val Cys Met Leu Gln Phe Pr - #o Ser Pro Ser Trp Tyr 195
- # 200 - # 205 - - Trp Asp Thr Val Thr Lys Ile Cys Val Phe Le - #u
Phe Ala Phe Val Val 210 - # 215 - # 220 - - Pro Ile Leu Ile Ile Thr
Val Cys Tyr Gly Le - #u Met Leu Leu Arg Leu 225 2 - #30 2 - #35 2 -
#40 - - Arg Ser Val Arg Leu Leu Ser Gly Ser Lys Gl - #u Lys Asp Arg
Ser Leu 245 - # 250 - # 255 - - Arg Arg Ile Thr Arg Met Val Leu Val
Val Va - #l Gly Ala Phe Val Val 260 - # 265 - # 270 - - Cys Trp Ala
Pro Ile His Ile Phe Val Ile Va - #l Trp Thr Leu Val Asp 275 - # 280
- # 285 - - Ile Asn Arg Arg Asp Pro Leu Val Val Ala Al - #a Leu His
Leu Cys Ile 290 - # 295 - # 300 - - Ala Leu Gly Tyr Ala Asn Ser Ser
Leu Asn Pr - #o Val Leu Tyr Ala Phe 305 3 - #10 3 - #15 3 - #20 - -
Leu Asp Glu Asn Phe Lys Arg Cys Phe Arg Gl - #n Leu Cys Arg Thr Pro
325 - # 330 - # 335 - - Cys Gly Arg Gln Glu Pro Gly Ser Leu Arg Ar
- #g Pro Arg Gln Ala Thr 340 - # 345 - # 350 - - Thr Arg Glu Arg
Val Thr Ala Cys Thr Pro Se - #r Asp Gly Pro Gly Gly 355 - # 360 - #
365 - - Gly Ala Ala Ala 370 - - - - (2) INFORMATION FOR SEQ ID
NO:11: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino -
#acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: - - Met Glu Leu
Val Pro Ser Ala Arg Ala Glu Le - #u Gln Ser Ser Pro 1 5 - # 10 - #
15 - - - - (2) INFORMATION FOR SEQ ID NO:12: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 amino - #acids (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:12: - - Cys His Pro Val Lys Ala Leu Asp Phe
Arg Th - #r Pro Ala Lys Ala Lys 1 5 - # 10 - # 15 - - Leu Ile Asn
Ile 20 - - - - (2) INFORMATION FOR SEQ ID NO:13: - - (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 16 amino - #acids (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:13: - - Leu Leu Ser Gly Ser Lys Glu Lys Asp
Arg Se - #r Leu Arg Arg Ile Thr 1 5 - # 10 - # 15 - - - - (2)
INFORMATION FOR SEQ ID NO:14: - - (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 16 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:14: - - Cys Gly Arg Gln Glu Pro Gly Ser Leu Arg Ar - #g Pro Arg
Gln Ala Thr 1 5 - # 10 - # 15 - - - - (2) INFORMATION FOR SEQ ID
NO:15: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino -
#acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: - - Cys Thr Pro
Ser Asp Gly Pro Gly Gly Gly Al - #a Ala Ala 1 5 - # 10
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